Virulent, wild-type human rotaviruses of major epidemiological importance were subjected to passage in cell culture at suboptimal temperature in an attempt to isolate cold-adapted mutants that were attenuated for humans and suitable for use in a live viral vaccine. Mutants of each of these rotaviruses were selected during successive serial passage in primary African green monkey kidney cells at progressively lower suboptimal temperature (30 degrees C, 28 degrees C, and 26 degrees C). The mutants exhibited both temperature sensitivity of plaque formation (i.e., a ts phenotype) and ability to form plaques efficiently at suboptimal temperature as compared to parental wild-type rotavirus (i.e., a cold-adaptation [ca] phenotype). A detailed analysis of viruses recovered after the individual passages in the 30 degrees C series indicated that the level of cold-adaptation observed at the end of the series was the result of two to three discrete incremental increases in ability to replicate and form plaques at 30 degrees C. The succeeding set of ten serial passages at 28 degrees C selected mutants that exhibited a greater degree of cold-adaptation and three of the mutants exhibited an associated increase in temperature sensitivity. Finally, in the case of three of the strains, the third successive serial passage series which was performed at 26 degrees C selected for mutants with an even greater degree of cold-adaptation that was associated with greater temperature sensitivity in one instance. This suggested that each of these viruses sustained a minimum of four to five mutations during the total selection procedure. These mutations imposed a varying degree of growth restriction at physiologic temperatures (i.e., 36 degrees -39 degrees C) in vitro. Recovery of mutants exhibiting a relatively wide range of growth restriction in vitro provides a collection of viruses that can be searched for promising live vaccine candidate strains. The availability of such a disparate set of mutants should increase the likelihood of identifying promising candidate vaccine mutants.